Proteomic analysis of hen egg white

Hen egg white is an original biological fluid in which major proteins have been widely studied, unlike the minor components. In this study, two-dimensional electrophoresis associated with mass spectrometry enabled the separation of 69 protein spots and their matching with major proteins, which were already known, and with minor proteins. Sixteen proteins were identified, and among them, two had never been previously detected in hen egg white, i.e., Tenp, a protein with strong homology with a bacterial permeability-increasing protein family (BPI), and VMO-1, an outer layer vitelline membrane protein. Thirteen proteins present a very wide polymorphism (ovotransferrin, ovomucoid, clusterin, etc.), some of them up to nine isoforms (ovoinhibitor). Eleven functional protein families were identified (serpin, transferrin, protease inhibitors Kazal, glycosyl hydrolases, lipocalin, bactericidal permeability-increasing protein, clusterin, UPAR/CD59/Ly6/ snake neurotoxin, cysteine protease inhibitor, VMO-1, and folate receptor families). These various biological functions could be interesting for further valorizations. In addition, three spots remain unidentified, probably because these proteins are not yet indexed in the international protein databanks.     

Dry-heating makes hen egg white lysozyme an efficient foaming agent and enables its bulk aggregation

Dry-heating is considered to be one of the most promising approaches to improving the functionality of food proteins. It has been shown that even if only minor structural modifications occur during dry-heating, the foaming properties of proteins are highly improved. With the recent results obtained in the field of foam stabilization by nanoparticles or protein aggregates in mind, a study was undertaken on the impact of dry-heating of lysozyme, used as a model protein, on its foaming properties. This work highlighted the fact that dry-heated hen egg white lysozyme simultaneously exhibited enhanced foaming properties and aggregation capacity. Although the conditions that favored bulk aggregation (high ionic strength, pH, treatment duration, and protein concentration) also favored foaming properties, the large bulk aggregates were not essential to obtain the best functionality. It is envisaged that heat-treated lysozyme may self-associate at the air/water interface, stabilizing air bubbles.     

Comparison of different electrophoretic separations of hen egg white proteins

The hen egg white protein composition has not yet been fully defined. To improve the knowledge of this biological fluid, the most usual and recently developed electrophoretic methods have been used: SDS-PAGE, native-PAGE, isoelectric focusing (IEF), and 2-dimensional electrophoresis (2DE). Seven of the major known proteins were thus identified in at least one electrophoretic system. Isoforms of ovotransferrin, ovalbumin, and ovomucoid were visualized when pI was used for the separation. Two-dimensional electrophoresis allowed separation of a very large number of spots. In each of the four systems, some components were revealed but not identified, and unknown spots were particularly numerous with 2DE. With this technique, many spots corresponding to small acidic proteins were highlighted, among which was the Ch21 protein, whose presence in hen egg white was thus confirmed. This study thus constitutes, to our knowledge, the first proteomic investigation of hen egg white.     

Identification and characterization of ovalbumin gene Y in hen egg white

Ovalbumin gene Y has been known as a member of the ovalbumin gene family since 1982, when its encoding gene was sequenced. In the present study, ovalbumin gene Y has been demonstrated as a new minor protein of hen egg white. This protein has been isolated by isoelectrofocalization and two-dimensional polyacrylamide gel electrophoresis and has been characterized using peptide mass fingerprinting. The concentration ratio of ovalbumin gene Y:ovalbumin is about 13:100. Unlike ovalbumin, ovalbumin gene Y is not phosphorylated, but like ovalbumin, this protein is glycosylated. Ovalbumin gene Y exists as a mixture of three molecular species, which differ in their isoelectric points. The polymorphism of this protein cannot be explained by various glycosylation levels.     

Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme

This work was aimed at the isolation, purification, and characterization of novel antimicrobial peptides from chicken egg white lysozyme hydrolysate, obtained by peptic digestion and subsequent tryptic digestion. The hydrolysate was composed of over 20 small peptides of less than 1000 Da, and had no enzymatic activity. The water-soluble peptide mixture showed bacteriostatic activity against Gram-positive bacteria (Staphylococcus aureus 23-394) and Gram-negative bacteria (Escherichia coli K-12). Two bacteriostatic peptides were purified and sequenced. One peptide, with the sequence Ile-Val-Ser-Asp-Gly-Asp-Gly-Met-Asn-Ala-Trp, inhibited Gram-negative bacteria E. coli K-12 and corresponded to amino acid residues 98-108, which are located in the middle part of the helix-loop-helix. Another novel antimicrobial peptide inhibited S. aureus 23-394 and was determined to have the sequence His-Gly-Leu-Asp-Asn-Tyr-Arg, corresponding to amino acid residues 15-21 of lysozyme. These peptides broadened the antimicrobial activity of lysozyme to include Gram-negative bacteria. The results obtained in this study indicate that lysozyme possesses nonenzymatic bacteriostatic domains in its primary sequence and they are released by proteolytic hydrolysis.     

Development of an ELISA for quantifying lysozyme in hen egg white

An indirect enzyme-linked immunosorbent assay (ELISA) by inhibition was developed for quantifying lysozyme in hen egg white (HEW), a protein of value in not only the food and pharmaceutical industries but also for poultry research. Various experimental conditions (coating, antibodies dilutions, samples dilutions, preparations, blocking agents, and incubation times) were assayed to optimize this assay to the quantification of HEW in egg white samples. HEW samples were diluted 1:3000 to avoid matrix effects, possibly resulting from lysozyme interaction with other egg white proteins. Assay linearity for lysozyme ranged from 0.38 to 4.8 mug/mL, with intra- and interassay variations of 6.8% and 7.6%, respectively, and the lower detection limit was 0.264 mug/mL. We found that lysozyme concentrations in albumen from eggs laid by a hen cohort ranged from 2.2 to 4.5 mg/mL, thus underlining interhen variability. Overall, these data present an ELISA assay that is simple, quick, sensitive, accurate, and has been specifically designed to determine lysozyme concentrations in egg white samples.     

Inhibiting effects of egg white dry-heated at 120 degrees C on heat aggregation and coagulation of egg white and characteristics of dry-heated egg white.

Dialyzed and freeze-dried egg white (FDEW) was dry-heated at 120 degrees C for up to 6 h. The inhibiting effects of the dry-heated egg white (DHEW) on the heat aggregation and coagulation of egg white (as 10% FDEW solution) and characteristics of the DHEW were examined. From the changes in turbidities and soluble protein contents of supernatant in various mixtures of 10% FDEW and DHEW solutions induced by heating (60 degrees C, 5 min), it was found that the inhibiting capacity increased with increases in the dry-heating time (DHT). The FDEW proteins were denatured with a mild conformational change (not secondary but tertiary structure) with the increase in DHT and aggregated partially. However, the more transparent solutions of DHEW containing soluble aggregates according to DHT were also obtained after heating. The transparency according to DHT came to be scarcely affected by the NaCl concentration and the dilution with diluents containing SDS, urea, and 2-mercaptoethanol. These findings suggest that the heat aggregations and coagulations of ovotransferrin and lysozyme in the FDEW were inhibited by their bindings with the soluble aggregates in DHEW.     

Enzyme immunoassay for hen egg white lysozyme used as a food additive

An indirect competitive enzyme immunoassay for hen egg white lysozyme (HEL) used as a food additive was investigated. Anti-HEL antibodies were obtained from B10A mouse ascites immunized by intraperitoneal injection of HEL. HEL samples to be assayed were extracted from foods with 1% gelatin in borate buffer. Goat anti-mouse IgG (H+L)-peroxidase complex was used as a second antibody, and 3,3',5,5'-tetramethylbenzidine was used as a substrate for the peroxidase. The working range for quantitative analysis was 1-50 ng/mL, because in this range the binding inhibition curve of anti-HEL antibodies to HEL-coated plates by HEL was linear. Even after losing the lysozyme activity by heat treatment, HEL could be detected by indirect competitive enzyme immunoassay. Recoveries of HEL by this assay were greater than 85% for Japanese noodles and Japanese traditional-style confectioneries, 53-95% for Miso and cooked beans, and 30-85% for fried fish pastes. HEL contents of 55 commercial foods were determined; HEL was detected in 19 samples in the range 25-20,000 ng/g. HEL as a food additive was detected more frequently in plant-derived foods than in foods of animal origin.     

Toward a better understanding of the lignin isolation process from wood

The recently developed protocol for isolating enzymatic mild acidolysis lignins (EMAL) coupled with the novel combination of derivatization followed by reductive cleavage (DFRC) and quantitative (31)P NMR spectroscopy were used to better understand the lignin isolation process from wood. The EMAL protocol is shown to offer access at lignin samples that are more representative of the overall lignin present in milled wood. The combination of DFRC/(31)P NMR provided a detailed picture on the effects of the isolation conditions on the lignin structure. More specifically, we have used vibratory and ball milling as the two methods of wood pulverization and have compared their effects on the lignin structures and molecular weights. Vibratory-milling conditions cause substantial lignin depolymerization. Lignin depolymerization occurs via the cleavage of uncondensed beta-aryl ether linkages, while condensed beta-aryl ethers and dibenzodioxocins were found to be resistant to such mechanical action. Condensation and side chain oxidations were induced mechanochemically under vibratory-milling conditions as evidenced by the increased amounts of condensed phenolic hydroxyl and carboxylic acid groups. Alternatively, the mild mechanical treatment offered by ball milling was found not to affect the isolated lignin macromolecular structure. However, the overall lignin yields were found to be compromised when the mechanical action was less intense, necessitating longer milling times under ball-milling conditions. As compared to other lignin preparations isolated from the same batch of milled wood, the yield of EMAL was about four times greater than the corresponding milled wood lignin (MWL) and about two times greater as compared to cellulolytic enzyme lignin (CEL). Molecular weight distribution analyses also pointed out that the EMAL protocol allows the isolation of lignin fractions that are not accessed by any other lignin isolation procedures.     

Metabolites of oxytetracycline, tetracycline, and chlortetracycline and their distribution in egg white, egg yolk, and hen plasma

4-epioxytetracycline and N-demethyloxytetracycline, as metabolites of oxytetracycline (OTC), 4-epitetracycline and N-demethyltetracycline, as metabolites of tetracycline (TC), and 4-epichlortetracycline, isochlortetracycline (ICTC), 4-epi-ICTC, and N-demethyl-ICTC, as metabolites of chlortetracycline (CTC), were detected in egg yolk and plasma obtained from feeding studies with either OTC, TC, or CTC. In egg white, only OTC, TC with its 4-epimer, and ICTC with its 4-epimer were detected in substantial concentrations. The ratios of epimerization and N-demethylation in the eggs did not change during the medication period. The samples were analyzed by an automated HPLC system (ASTED) with UV, fluorescence, or MS-MS detection.     

Stability of acidic egg white protein emulsions containing xanthan gum

The influence of xanthan gum concentration on the physicochemical stability of model oil-in-water emulsions prepared with egg white protein at pH 3.8 and containing 150 mM NaCl was investigated by following droplet aggregate formation, rheological changes, and serum separation with storage time. Egg white emulsions were more strongly flocculated and exhibited higher stability against creaming than those of yolk, irrespective of the presence or absence of xanthan. Depletion effects, originating from the presence in the continuous phase of the emulsions of nonadsorbing xanthan molecules, intensified droplet-droplet flocculation effects and resulted in large droplet flocs. At relatively low xanthan contents, the emulsions exhibited higher stability against creaming compared to the respective control emulsions probably due to the formation of a continuous droplet aggregate network structure. At higher xanthan contents, less extensive droplet interactions, due to slowly evolving microstructure of phase-separated xanthan-rich and xanthan-depleted regions, resulted in emulsions exhibiting increased stability against creaming. The role of interactions between protein molecules adsorbed on neighboring droplets in these changes and their effect on emulsion aging are discussed.     

Effect of cholecalciferol-enriched hen feed on egg quality

Eggs are one of the most important sources of vitamin D in the human diet, and their vitamin D content can be further increased by adding more vitamin D to hen feed. To investigate this issue more closely, we performed two feeding experiments. In both, zero egg samples were collected while the hens were fed regular feeds with a vitamin D content of 1720 or 4280 IU/kg. In experiment 1, egg samples were collected 2, 4, 7, 9, 11, 13, 16, 23, and 30 days after beginning the high-cholecalciferol (11 200 IU/kg) feeding period. In experiment 2, samples were collected 2, 4, 6, 8, 13, 28, 56, 84, 112, 140, and 168 days after beginning the high-cholecalciferol (12 000 IU/kg) diet. The egg samples were then assayed for their cholecalciferol content, and some samples, also for the presence of 25-hydroxycholecalciferol by an HPLC method. Further, the vitamin D-fortified eggs were compared with the controls by a sensory evaluation, by conducting fatty acid and functional analyses (emulsion capacity, gel forming capacity, foaming properties) and by measuring eggshell strength. Because vitamin D can be toxic in high doses, we also performed histopathological tests on the hens at the end of experiment 2. The top cholecalciferol contents in egg yolk (ca. 30 microg/100 g) were reached 8-13 days from starting the high-cholecalciferol diet. After 112 days feeding the cholecalciferol content gradually decreased to ca. 22 microg/100 g. When added to eggs as described above, vitamin D did not affect their sensory or functional properties or their fatty acid composition. Moreover, the cholecalciferol levels used in this study appeared not to affect eggshell strength or to be harmful for hens.     

Characterization of heat-induced changes in skim milk using asymmetrical flow field-flow fractionation coupled with multiangle laser light scattering

Separation and size measurement of protein particles are a relevant approach to monitor heat-induced changes in skim milk. Unfortunately, no method is currently available at low cost and without excessive preparation of the samples. Therefore, the present study aimed at evaluating the interest of asymmetrical flow field-flow fractionation (AFlFFF) coupled with multiangle laser light scattering (MALLS) for this purpose. Unheated and heated skim milk samples at pH 6.5 and 7.2 were prepared and comparatively analyzed using AFlFFF-MALLS, size exclusion chromatography (SEC-MALLS) and dynamic light scattering. The results showed that AFlFFF could evidence the conversion of the native whey proteins of unheated milk into heat-induced whey protein/κ-casein complexes in the serum phase of milk and possibly on the surface of the casein micelles. The pH-induced changes in the partition of the complexes between the serum and the micellar phases could also be observed. The results therefore showed the interest of AFlFFF-MALLS to monitor the heat-induced changes in particle sizes in skim milk and to separate the different protein components of unheated and heated skim milk.     

Isolation and characterization of immunoglobulin in yolk (IgY) specific against hen egg white lysozyme by immunoaffinity chromatography

Six hens were intramuscularly (im) immunized once a week for 3 weeks using chicken egg white lysozyme (LS) as antigen. Antibody (immunoglobulin in yolk, IgY) ELISA values of 10(3)-fold diluted yolk were almost as high as 1.879 in the sixth week and maintained a value of 0.756 in the eighth week after the initial immunization treatment. The purification efficiency (specific activity of purified IgY against LS/specific activity of antibody in yolk against LS) of IgY specific against LS isolated by laboratory-prepared LS-bound (IgY-) Sepharose 4 Fast Flow immunoaffinity column was approximately 3380. By applying various amounts (0-22 mg) of the thusly obtained IgY specific against LS to the immunoaffinity column, the binding capacity (q(m)) and dissociation constant (K(d), M(-1)) of such immunoaffinity gel for IgY against LS were found to be 0.68 mg of IgY/mL of wet gel (0.54 mg of IgY/mg of LS) and 7.13 x 10(-6) M, respectively, as determined by Langmuir-type adsorption isotherms.     

Emulsifying and foaming properties of ultraviolet-irradiated egg white protein and sodium caseinate

The physicochemical and functional properties of ultraviolet (UV)-treated egg white protein (EW) and sodium caseinate (SC) were investigated. UV irradiation of the proteins was carried out for 30, 60, 90, and 120 min. However, the SC samples were subjected to extended UV irradiation for 4 and 6 h as no difference was found on the initial UV exposure time. Formol titration, SDS-PAGE, and FTIR analyses indicated that UV irradiation could induce cross-linking on proteins and led to improved emulsifying and foaming properties (P < 0.05). These results indicated that the UV-irradiated EW and SC could be used as novel emulsifier and foaming agents in broad food systems for stabilizing and foaming purposes.     

重力场流分离结合图像处理测定小麦粉粒径的研究

针对小麦粉颗粒大小的测量问题，首先利用重力场流分离方法对小麦粉进行了分离。在分离基础上利用图像处理方法完成了对小麦粉粒径的测量。经重力场分离后，小麦粉样本被分为颗粒大小相近的3部分。试验中使用光学显微镜及CCD摄像头评定分离效果。试验结果表明试验样本小麦粉的粒径主要分布范围在17.0～18.5、6.7～8.0、2～3.5 μm三个尺寸段内。该研究为进一步研究小麦粉的特性提供了一种全新的手段。     

Correlation of the protein structure and gelling properties in dried egg white products

The relationship between protein structure and aggregation, as well as heat-induced gelling properties, of seven dried egg white (DEW) products was investigated. Strong correlations were found between average molecular weight and hydrophobicity plus surface SH groups of DEW-soluble protein aggregate (SPA). This suggests that hydrophobic interactions and disulfide bond formation between protein molecules were involved in the aggregation. The average molecular weight of DEW products with alkaline pHs was relatively higher than those with neutral pHs and the same degree of protein unfolding, probably because of more disulfide bond formation between protein molecules. In addition, strong correlations were found between hydrophobicity, surface SH groups plus average molecular weight of DEW-SPA, and physical properties of the gels from DEW products. These data indicated that controlling the aggregation of DEW proteins in the dry state is crucial to controlling the gelling properties of DEW.     

Cross-linking of hen egg white lysozyme by microbial transglutaminase under high hydrostatic pressure: localization of reactive amino acid side chains

After incubation of hen egg white lysozyme (HEWL) with microbial transglutaminase (mTG) under high pressure (400-600 MPa for 30 min at 40 °C), the formation of HEWL oligomers was observed via SDS electrophoresis. At atmospheric pressure, HEWL represents no substrate for mTG. Likewise, enzymatic treatment following a pretreatment with high pressure did not lead to oligomerization. Reactive amino acid side chains were identified by peptide mapping after tryptic digestion using RP-HPLC with ESI-TOF-MS. Isopeptide-containing peptide fragments were found only in HEWL samples simultaneously treated with enzyme and pressure. It was found that mTG exclusively cross-links HEWL under high pressure by formation of an isopeptide between lysine at position 1 and glutamine at position 121 in the peptide chain. Therefore, a pressure-induced partial and reversible unfolding of the protein with exposure of lysine and glutamine side chains has to occur, resulting in a site-directed oligomerization of HEWL by mTG. The enzymatic modification of HEWL by mTG under high pressure offers interesting perspectives for further functionalization reactions.     

Identification of an IgE reactive peptide in hen egg riboflavin binding protein subjected to simulated gastrointestinal digestion

Riboflavin binding protein (RfBP) is a minor protein in hen egg; its potential involvement in egg allergy has seldom been studied. The aim of this work was to investigate the IgE binding capacity of RfBP before and after simulated gastrointestinal digestion. It was shown that digestion of RfBP mainly occurred during the gastric phase. The protein fragments resulting from the subsequent duodenal phase remained linked through disulfide bonds. Both the intact protein and its digests were subjected to inhibition ELISA with sera obtained from patients allergic to egg. The results revealed significant IgE binding to intact RfBP, whereas the digests showed reduced but substantial IgE binding levels, with serum-to-serum variability. The RfBP digests were then subjected to immunoblot with allergic patients' sera, and the IgE-reactive peptides were further analyzed by MALDI-TOF/TOF mass spectrometry for sequence determination. The RfBP sequence 41-84 was identified as a novel IgE binding peptide in patients allergic to egg.